Buprenorphine, N-cyclopropylmethyl-7α-[1-(5)-hydroxy-1,2,2-trimethylpropyl]-6,14-endo-ethano-6,7,8,14-tetrahydronoripavine, is a highly lipophylic opiate analog with both agonist and antagonist properties at the μ-opiate receptor (Martin et al., J. Pharmacol. Exp. Ther., 197:517 (1976); Cowan et al., Br. J. Pharmacol., 60: 537 (1977); Heel et al., Drugs, 17:81 (1979)). As an analgesic, it has a potency about 30 times higher than that of morphine and 75 times higher than that of pentazocine when administered intravenously or intramuscularly and is effective in the treatment of acute and chronic pain. Buprenorphine has a long duration of action because it dissociates slowly from the buprenorphine-receptor complex (Hambrook et al., Opiates and Endogenous Opiate Peptides, 295–301 (1976)). Another important feature of the drug is its limited effect on respiration, even in overdose (Hand et al., Ann. Clin. Biochem., 23: 47–53 (1986); Banks et al., N.Z. Med. J., 89: 256–257 (1979)). Due to its potency and other beneficial properties, buprenorphine is used broadly for pain management in, for example, cancer and postoperative patients.
Further, due to its long duration of action, its ability to antagonize opiates, its low dependence liability and lack of significant withdrawal symptoms compared to heroin, cocaine, and other narcotics, buprenorphine is also useful in the management of opiate dependency, including the rehabilitation of opiate addicts (Jasinski et al., Arch. Gen. Psychiatry, 35:501 (1978); Mello et al., Science, 207: 657 (1980)). Nevertheless, despite its lower physical dependence liability, buprenorphine abuse has been reported (Strang, Lancet, 25: 725 (1985); Robertson et al., Br. Med. J., 292: 1465 (1986); Chowhurdy et al., Br. J. Addiction, 85: 1349 (1990)).
The chemistry of buprenorphine metabolism in man and other animals (e.g., canine and equine) has been well-studied, with the kinetics reported in plasma (Hand et al., Ann. Clin. Biochem., 23: 47–53 (1986); McQuay et al., Advances in Pain Research and Therapy, pp. 271–278 (1986); Bullingham et al., Clin. Pharmacokinet., 8: 332–343 (1983)) and in urine (Cone et al., Drug Metab. Dispos., 12: 577–581 (1984); Hand et al., J. Anal. Tox., 13: 100–104 (1989); Heel et al., Drugs, 17: 81 (1979)) after intravenous, intramuscular, and sublingual administration. The parent drug is nearly completely metabolized to norbuprenorphine, norbuprenorphine 3-O-β-D-glucuronide (norbuprenorphine glucuronide), and buprenorphine 3-O-β-D-glucuronide (buprenorphine glucuronide) and is consequently present in urine at extremely low concentrations. After sublingual administration, buprenorphine plasma concentrations rise slowly and are maintained at low concentrations for several hours (Bullingham et al., Br. J. Clin. Pharmacol., 13: 665–673 (1982)). Buprenorphine has a long half-life of about 8 hours, and the norbuprenorphine metabolite appears to have an even slower elimination (Hand et al., Ann. Clin. Biochem., 23: 47–53 (1986); McQuay et al., Advances in Pain Research and Therapy, pp. 271–278 (1986); Bullingham et al., Clin. Pharmacokinet., 8: 332–343 (1983)). This accords with the appearance of buprenorphine and its buprenorphine glucuronide metabolite in urine in 1–2 days and norbuprenorphine and norbuprenorphine glucuronide in 1–4 days (Cone et al., Drug Metab. Dispos., 12: 577–581 (1984); Blom et al., J. Chromatogr., 338: 89–98 (1985)). Heel et al. reports that approximately 15–27% of a dose of buprenorphine appears in the urine, mainly in the form of glucuronide metabolites of the parent compound and the norbuprenorphine metabolite (Heel et al., Drugs, 17:81 (1979)).
The accurate detection of buprenorphine and its metabolites in a biological sample, such as plasma or urine, is useful for several purposes, including determining the illicit use or abuse of buprenorphine, monitoring the dose and efficacy of buprenorphine during clinical treatment for pain, and confirming the prescriptive use of buprenorphine, for example, confirming its use in a drug rehabilitation program. Furthermore, given that the criteria of assay performance for one use may not be applicable for other uses, the differential detection of buprenorphine and its metabolites, i.e., an assay that distinguishes between buprenorphine and buprenorphine metabolites, is useful. For example, confirmation of appropriate rehabilitative use of buprenorphine requires the accurate detection of buprenorphine metabolites rather than buprenorphine in urine, as the presence of significant amounts of the unmetabolized parent drug in a urine sample indicates patient tampering, i.e., adulteration of the sample with buprenorphine to feign compliance with the rehabilitation program. Thus, an assay for the sensitive detection of a buprenorphine metabolite is required to accurately confirm rehabilitative use of buprenorphine. In some cases, it is critical to accurately measure only the parent drug. For example, when buprenorphine is used for the management of pain, only plasma levels of the parent drug are relevant (D. Moody et al., J. Anal. Toxicol. 21:406–414, 1997). In other instances, it is appropriate to measure either the parent drug or its metabolites, or both the parent drug and its metabolites. For example, the determination of buprenorphine abuse in humans and other mammalian subjects can be made by detecting the presence of buprenorphine and/or one or more of its metabolic products.
Current methods for the detection of buprenorphine and its metabolites include chromatographic methods, including thin layer chromatography, gas chromatography and high-performance liquid chromatography, which can be used to detect the parent drug buprenorphine (Hackett et al., J. Chromatography, 374: 400–404 (1986)). However, these methods can be time-consuming and expensive and can also lack the sensitivity required for the accurate quantitation of buprenorphine and/or its metabolites.
More recently, fluorometric or radiometric immunoassays employing the use of a polyclonal antisera that binds to buprenorphine and/or its metabolites have also been used for the detection of these compounds in biological samples. However, current immunoassays are limited by the sensitivity and/or cross-reactivity of the polyclonal antisera used. Cross-reaction of antisera with metabolites can cause lack of specificity in the measurement of buprenorphine in the absence of extraction steps to overcome metabolite interference, which may result in an overestimate of buprenorphine concentration (Bartlett et al., Eur. J. Clin. Pharmacol., 18: 339–345 (1980); Debrabandere et al., Analyst, 118:137–143 (1993)). Debrabandere et al. describes a polyclonal antibody that cross-reacts with buprenorphine and norbuprenorphine such that it can not distinguish between the parent drug and the metabolite (Debrabandere et al., Analyst, 118:137–143 (1993)) Further, the polyclonal antibody fails to recognize the buprenorphine glucuronide metabolite.
Other groups have described the use of polyclonal antibodies cross-reactive with buprenorphine and a specific metabolite thereof, i.e., norbuprenorphine or buprenorphine glucuronide (Bartlett et al., Eur. J. Clin. Pharmacol., 18: 339–345 (1980); Hand, et al., Ann. Clin. Biochem., 23: 47–53 (1986); Hand et al., J. Anal. Tox., 13: 100–104 (1989)). As is the case generally, the above-mentioned polyclonal antibodies were raised by immunizing a mammal with a conjugate of the particular buprenorphine metabolite of interest (or a derivative thereof). The resulting polyclonal antibodies demonstrate a limited cross-reactivity to other metabolites, as well as to buprenorphine, relative to the particular metabolite used for immunization. Thus, the sensitivity of the polyclonal antibodies for buprenorphine is diminished. Furthermore, producing a polyclonal antibody specific for a particular buprenorphine metabolite can be difficult given that it requires procurement of a sufficient amount of the specific metabolite for preparation of an immunogen conjugate, which metabolite may not be commercially available and may be burdensome to synthesize.